Regulation of gene expression underlies disparate biological processes, including development of an organism from a fertilized egg, the response of organisms to extra-cellular signals, and the ingraining of memories. The grant requests funds to continue our analyses of mechanisms of gene regulation that will help us understand the normal processes and shed light on how they can go awry. The proposed studies use primarily yeast cells, a eukaryote in which many aspects of gene regulation are closely related to gene regulation in mammalian cells. In a series of papers and books we have developed a model - called the 'recruitment'model - for how transcriptional regulators determine whether any given gene will be activated or repressed. We propose here a series of experiments that probe and test this model in various ways. We have developed methods to measure the appearance, at a specific gene, of various protein complexes as they are recruited by a DNA-bound activator. Over 100 proteins must be brought to the gene for transcription to proceed, and we are using our methods to study how this large complex is formed stepwise. In a related development, we now can assay with great accuracy the disposition of nucleosomes on DNA, and we are studying how nucleosome positioning and removal is effected by other DNA binding proteins. For reasons that are not understood, a transcriptional activator can turn on transcription of a gene when bound many hundreds of base pairs from the gene in higher eukaryotes, but in yeast the activator must be bound much closer to the gene to work efficiently. We have modified yeast so as to allow 'activation at a distance', and we are now analyzing the mechanism to see whether the effect is consistent with the recruitment model. And we are examining the properties of a protein that seems to have a unique ability to trigger gene expression when artificially recruited to a gene. In particular we are testing the idea that this protein, a component of the large complex called the Mediator, is rapidly degraded in cells, and that this instability facilitates activation of transcription